Elimination of targeted cell populations with immunopharmaceuticals is an important therapeutic intervention in several indications. The mechanisms of action used by immunopharmaceuticals to effect such elimination of targeted cells can include complement mediated cellular lysis, activation of apoptotic signaling pathways, blockade of signaling pathways required for survival, and antibody-dependent cellular cytotoxicity (ADCC), also referred to as Fc-dependent cellular cytotoxicity. ADCC is a potent mechanism that is believed to be important for the efficacy of many immunopharmaceuticals.
The mechanism for activation of ADCC involves binding of Fc receptors to immunopharmaceutical molecules that are bound to the surface of the target cell. The binding of Fc receptors to immunopharmaceuticals can be mediated by domains within the constant region of immunoglobulins, such as the CH2 and/or CH3 domains. Different types of constant regions may bind different Fc receptors. Examples include the binding of IgG1 Fc domains to cognate Fc receptors CD16 (FcγRIII), CD32 (FcγRII-B1 and -B2), and CD64 (FcγRI), IgA Fc domains to the cognate Fc receptor CD89 (FcαRI), and IgE domains to cognate Fc receptors FcεFR1 and CD23.
Immunopharmaceutical compositions with enhanced Fc receptor binding may exhibit greater potency in ADCC. Reported methods of achieving this with IgG Fc domains include the introduction of amino acid changes and the modification of carbohydrate structures. Modification of carbohydrate structures may be preferable as amino acid changes in the Fc domain may enhance immunogenicity of a pharmaceutical composition. For immunoglobulin molecules it has been demonstrated that attachment of N-linked carbohydrate (oligosaccharide) to Asn-297 of the CH2 domain is critical for ADCC activity. Its removal enzymatically or through mutation of the N-linked consensus site results in little to no ADCC activity. Some studies have reported that the level of ADCC activity for an immunoglobulin molecule is also dependent on the structure of the carbohydrate, but the actual carbohydrate moieties or structure responsible for ADCC have not yet been elucidated. Still less is known about the optimal carbohydrate structure(s) for ADCC of non-immunoglobulin Fc fusion proteins.
In glycoproteins, carbohydrates may attach to the amide nitrogen atom in the side chain of an asparagine in a tripeptide motif Asn-X-Thr/Ser. This type of glycosylation, termed N-linked glycosylation, commences in the endoplasmic reticulum (ER) with the addition of multiple monosaccharides to a dolichol phosphate to form a 14-residue branched carbohydrate complex. This carbohydrate complex is then transferred to the protein by the oligosaccharyltransferase (OST) complex. Before the glycoprotein leaves the lumen of the ER, three glucose molecules are removed from the 14-residue oligosaccharide. The enzymes ER glucosidase I, ER glucosidase II and ER mannosidase are involved in ER processing.
Subsequently, the polypeptides are transported to the Golgi complex, where the N-linked sugar chains are modified in many different ways. In the cis and medial compartments of the Golgi complex, the original 14-saccharide N-linked complex may be trimmed through removal of mannose (Man) residues and elongated through addition of N-acetylglucosamine (GlcNac) and/or fucose (Fuc) residues. The various forms of N-linked carbohydrates generally have in common a pentasaccharide core consisting of three mannose and two N-acetylglucosamine residues. Finally, in the trans Golgi, other GlcNac residues can be added, followed by galactose (Gal) and a terminal sialic acid (Sial). Carbohydrate processing in the Golgi complex is called “terminal glycosylation” to distinguish it from “core glycosylation,” which takes place in the ER. The final complex carbohydrate units can take on many forms and structures, some of which have two, three or four branches (termed biantennary, triantennary or tetraantennary). A number of enzymes are involved in Golgi processing, including Golgi mannosidases IA, IB and IC, GlcNAc-transferase I, Golgi mannosidase II, GlcNAc-transferase II, Galactosyl transferase and Sialyl transferase.
One report has suggested that a crucial carbohydrate determinant of FcγRIIIa receptor-mediated ADCC activity is the lack of an alpha-1,6-fucose moiety added to the core N-linked structure (Shinkawa et al., J Biol Chem. 2003 Jan. 31; 278(5):3466-73; see also Shields et al., J Biol Chem. 2002 Jul. 26; 277(30):26733-40). The level of another glycoform, bisected N-linked carbohydrate, has also been proposed to be capable of imparting increased ADCC (Umana et al., Nat. Biotechnol. 1999 February; 17(2): 176-80) but there is also contradictory evidence (Shinkawa et al., J Biol Chem. 2003 Jan. 31; 278(5):3466-73). A potential solution to this contradictory evidence has been suggested by the finding that increased GnTIII in host cells produces immunoglobulin not only with increased bisected sugar but also lacking the core fucose modification (Ferrara et al., Biotechnol Bioeng. 2006 Apr. 5; 93(5):851-61). This agrees with suggestion that fucose alone has the key role in altering ADCC potency and the association with bisected sugar seen by others reflects a linkage in the two modifications in host cells. However, another report in which in vitro treatment of Rituxan and Herceptin antibodies with GnTIII, to increase bisected sugar, resulted in increased ADCC suggests a direct effect of bisected sugar (Hodoniczky et al., Biotechnol. Prog., 2005 November-December 21(6): 1644-52). However, overexpression of Gnt III at very high levels may be toxic to the cell (Umana et al., Biotechnol Prog. 1998 March-April; 14(2):189-92).
Some proposed methods for producing immunoglobulins with lower fucose content have significant drawbacks for manufacture of a biopharmaceutical drug with an optimal ADCC activity for the therapeutic indication. For example, treatment of immunoglobulins with enzymes that remove fucose residues (fucosidases) involves additional costly manufacturing steps with potentially significant economic and drug consistency risks. Molecular engineering of cell lines to knock-out key enzymes involved in the synthesis of fucosylated glycoproteins require special host strains and in current practice do not allow for “tunable” production of drug with varying ADCC potency to optimize efficacy and safety for a therapeutic use. Generation of a comparison non-enhanced ADCC product is expensive and time consuming. The treatment of cell lines with RNAi or antisense molecules to knock down the level of these key enzymes may have unpredictable off-target effects and would be costly if not impractical to implement at manufacturing scale.
Thus, there continues to exist a need for advantageous methods of preparing immunopharmaceuticals with enhanced ADCC as well as for the improved immunopharmaceuticals produced thereby for therapeutic uses.